Soybean seeds and plants exhibiting natural herbicide resistance

ABSTRACT

Soybean plants are provided that exhibit in the absence of induced mutagenesis naturally-occurring genetically-controlled glyphosate herbicide resistance that is not attributable to genetic engineering. Mature soybean seeds initially are soaked in a liquid comprising a glyphosate herbicide for a period of time sufficient for the herbicide to reach the embryos of the soybean seeds. Following such soaking the soybean seeds are planted to produce at least one soybean plant or a descendant thereof in a subsequent generation that displays resistance to glyphosate herbicide. The existence of the naturally-occurring glyphosate herbicide resistance in a soybean plant is confirmed by demonstrating the absence of a foreign gene for herbicide resistance that has been introduced by genetic engineering. Soybean seeds capable of forming the naturally-occurring genetically-controlled glyphosate herbicide resistance that is not attributable to genetic engineering also are provided. Such herbicide resistance can be reliably expressed and transferred to other soybean plants by conventional plant breeding methods.

CROSS-REFERENCE TO RELATED APPLICATION

This is a Continuation-in-Part of U.S. patent application Ser. No.10/119,194, filed Apr. 10, 2002.

FIELD OF THE INVENTION

The invention provides a process for selecting soybean plants andgenerating soybean lines that exhibit naturally-occurringgenetically-controlled herbicide resistance in the absence of geneticengineering, soybean lines that exhibit naturally-occurring herbicideresistance, the naturally-occurring genes that confer such herbicideresistance that are exposed by the aforementioned selection process, andthe use of such herbicide resistance genes through genetic engineeringto confer herbicide resistance to other plants that are naturallysensitive to herbicide exposure.

BACKGROUND OF THE INVENTION

Soybean plants of the genus Glycine max L. have long been recognized tobe an important crop, which is being grown in many parts of the world.This crop is grown primarily for the seeds that are produced. Theseseeds may be used for planting or as a source of edible and industrialoils with the residue serving as a livestock feed supplement.

Modern agriculture practices are increasingly taking advantage ofherbicides to eliminate unwanted weeds from soybean fields and tominimize the expense of tilling fields to remove unwanted weeds.Presently, there are no herbicides that kill on contact (post-emergentherbicides) that can be used with conventional soybean plants withoutcausing excessive crop injury. The herbicide glyphosate is an effectivenon-selective post-emergent herbicide. Plant transformation/geneticengineering has been used in the past to modify soybean plants toincorporate resistance to the herbicidal effects of glyphosate.

Genetic engineering/plant transformation involves the incorporation of agene for herbicide resistance into the chromosome of the soybean plant.Such procedures require special expertise and can be very costly. The“resistance” gene is part of a construct that is placed in the plant toimpart herbicide resistance. In addition, the construct containspromoters that are responsible for activating the gene in selectportions or in all parts of the plant. The presence or absence of thesepromoters is used to determine if the plant is the result of geneticengineering/plant transformation. The gene construct that is incommercially available in soybean plants includes the promoters,CaMV35S, and NOS marker gene. Representative prior publications thatconcern the use of genetic engineering to produce such herbicideresistance include U.S. Pat. Nos. 4,971,908; 5,145,783; 5,312,910;5,352,605; 5,530,196; and 5,858,742.

It also has been proposed in the past to attempt to create somesulfonylurea herbicide resistance in soybean plants through the use ofinduced mutagensis. See, for instance, (a) “Molecular Strategies forCrop Protection”, by R. S. Chaleff et al., Pages 415 to 425 (1987), (b)“Soybean Mutants with Increased Tolerance for Sulfonylurea Herbicides”,by Scott A. Sebastian et al. Crop Science, 27, Pages 948 to 952 (1987),and (c) “Semidominant Soybean Mutation for Resistance to SulfonylureaHerbicides”, S. A. Sebastian et al., Crop Science, 29, Pages 1403 to1408 (1989). The resulting plants display no glyphosate herbicideresistance, often display limited resistant to sulfonylurea herbicide,and such herbicide is sometimes ineffective to kill weeds in view ofacquired resistance to the sulfonylurea herbicide.

The objective of the present invention is to provide a new and reliableroute for providing genetically-controlled herbicide resistance insoybean plants in the absence of induced mutagensis or the insertion ofa foreign gene. A further objective of the present invention is toprovide a soybean plant having genetically-controlled resistance thatcan be contacted with the herbicide in all stages of the life cycle ofthe plant and seeds capable of producing the same. The invention alsoprovides a soybean plant that contains within its genome a naturallyoccurring genetic determinant that confers herbicide resistance that canbe genetically mapped and physically isolated for use in breeding orbiotechnological programs. Such genetic determinants represent novelgenetic mechanisms for conferring herbicide resistance to host plants.

These and other objectives, as well as the scope, nature and utilizationof the claimed invention will be apparent to those skilled in this areaof technology from the following detailed description and appendedclaims.

SUMMARY OF THE INVENTION

The invention provides a method for detecting, isolating, and developingsoybean plants that exhibit naturally-occurring genetically-controlledherbicide resistance in the absence of genetic engineering/planttransformation. The method comprises soaking mature soybean seeds in aliquid comprising a herbicide for a period of time sufficient for theherbicide to reach the embryos of the soybean seeds, planting saidsoybean seeds following said soaking in a growing medium and producingat least one soybean plant that displays resistance to the herbicide,selecting a soybean plant which exhibits genetically-controlledherbicide resistance that is not attributable to a foreign gene forherbicide resistance introduced by genetic engineering and is the resultof the action of a naturally-occurring herbicide resistance gene. In apreferred embodiment the herbicide is glyphosate.

The invention also provides a method for selecting individual soybeanplants that are herbicide resistant from soybean germplasm or soybeanlines identified as described in the method identified above. Thismethod entails planting large numbers of seeds from soybean germplasm orsoybean lines identified using the method described above, sprayingplants with liquid herbicide solutions at herbicide concentrationssufficient to kill soybean plants and weeds, selecting survivors fromthe herbicide treatments and selecting soybean plants which exhibitnaturally-occurring genetically-controlled herbicide resistance that isnot attributable to a foreign gene for herbicide resistance introducedby genetic engineering from the survivors.

The invention also provides the isolation of seeds from the individualherbicide resistant soybean plants identified using the method describedabove.

The invention also provides the establishment of plants derived from theseeds described above.

The invention also provides the isolation of the nucleic acids thatdirect natural herbicide resistance from the genome of the soybeanplants isolated using the methods described herein.

The invention also provides DNA constructs comprising the nucleic acidsas described. In such constructs the nucleic acid is operatively linkedto plant gene expression control sequences.

The invention also provides vectors comprising the DNA construct asdescribed herein.

The invention further provides a transgenic plant or part of a plant.The transgenic plant or part of a plant comprises the nucleic acidoperatively linked to plant gene expression control sequences producedaccording to the present invention.

The invention also provides a method of controlling weeds in a fieldcontaining herbicide resistant soybean plants of the present inventionor transgenic plants utilizing nucleic acid sequences, constructs orvectors formed in accordance with the present invention.

The invention further provides the following:

A soybean plant or seed of the line NatGen 1 or its descendants havinggenetically-controlled glyphosate herbicide resistance that isattributable to the homozygous gene NG^(R1)NG^(R1) obtainable from ATCCAccession No. PTA-4774.

A soybean plant or seed of the line NatGen 2 or its descendants havinggenetically-controlled herbicide resistance that is attributable to thehomozygous gene NG^(R2)NG^(R2).

A soybean plant or seed of the line NatGen 3 or its descendants havinggenetically-controlled herbicide resistance that is attributable to thehomozygous gene NG^(R3)NG^(R3) obtainable from ATCC Accession No.PTA-5937.

A soybean plant or seed of the line NatGen 4 or its descendants havinggenetically-controlled herbicide resistance that is attributable to thehomozygous gene NG^(R4)NG^(R4).

A soybean plant or seed of the line NatGen 5 or its descendants havinggenetically-controlled herbicide resistance that is attributable to thehomozygous gene NG^(R5)NG^(R5).

A soybean plant or seed of the line NatGen 6 or its descendants havinggenetically-controlled herbicide resistance that is attributable to thehomozygous gene NG^(R6)NG^(R6).

A soybean plant or seed of the line NatGen 7 or its descendants havinggenetically-controlled herbicide resistance that is attributable to thehomozygous gene NG^(R7)NG^(R7) obtainable from ATCC Accession No.______.

Isolated nucleic acids comprising of any of the NG^(R1-R7)NG^(R1-R7)genes, or gene systems, and expression controlling elements derived fromsoybean plants are made possible which when expressed in a soybeanplants, other dicotyledonous crops, or monocotyledonous crops (aftersuitable modification to the coding sequence and/or controlling elementsthat are standard expedients to one skilled in art) render that plantand its progeny resistant to the herbicide.

DESCRIPTION OF PREFERRED EMBODIMENTS

Presently useful herbicide resistance in commercial soybean varieties islimited to only a few herbicides, the most important one beingglyphosate. Resistance to glyphosate has been achieved by theintroduction of bacterial genes into soybean germplasm that either codesfor target enzymes that are not affected by the herbicide or for enzymesthat break down the herbicide into an inactive form. Such genes havebeen constructed and introduced into soybean plants using standardgenetic engineering techniques of gene construction and planttransformation. Examples of such genes are described in U.S. Pat. Nos.4,971,908; 5,145,783; 5,312,910; 5,352,605; 5,530,196; and 5,858,742.These genes are engineered to function in a plant cell and are placedunder the control of a promoter element, commonly derived from a plantviral genome that allows for the constitutive expression of theherbicide resistant or degradative enzyme. Such biotechnologicalstrategies demand a lengthy and expensive research and developmentprogram. The present invention provides a method by whichnaturally-occurring genes that confer herbicide resistance and that arealready present in soybean germplasm stocks can be revealed, identifiedand exploited for commercial use in both conventional andbiotechnological breeding programs.

The initial step of the process of the present invention soybean seedsare soaked in a liquid comprising a herbicide. Commonly the herbicide ispresent in concentrations that are sufficient to kill conventionalsoybean plants as well as weeds that commonly occur in soybean fields.Representative herbicides are glyphosate, 2,4-dichlorophenoxyaceticacid, glufosinate ammonium butanoic acid,3,5-dibromo-4-hydroxybenzonitrile, etc. In a preferred embodiment theherbicide is a glyphosate and the herbicide concentration is sufficientto kill soybean plants at the commercially used rate of 1 quart per acreof a glyphosate preparation that is 41% active ingredient(N-(phosphonomethyl)glycine. Such herbicide isN-(phosphonomethyl)glycine of the chemical formula:

and is commercially available from Monsanto Corporation under theROUNDUP trademark as well as from other companies under varioustrademarks. This herbicide is a non-selective, broad spectrum,post-emergence herbicide that is registered for use in more than fiftycrops. This molecule is an acid, which dissociates in aqueous solutionto form phytoxic anions. Several anionic forms are known. As usedherein, the name “glyphosate” refers to the acid and its anions.Glyphosate inhibits the shikimic acid pathway that provides a precursorfor the synthesis of aromatic amino acids. Specifically, glyphosatecurbs the conversion of phosphoenolpyruvate and 3-phosphoshikimic acidto 5-enolpyruvyl-3-phospho shikimic acid by inhibiting the enzyme5-enolpyruvyl-3phosphoshikimiate synthase. Although glyphosate isidentified in this preferred embodiment, other herbicides, such as2,4-dichlorophenoxyacetic acid, glufosinate ammonium butanoic acid, or3,5-dibromo-4-hydroxybenzonitrile, etc., can be used to identify andisolate herbicide resistance genes present in the soybean germplasm.

Preferably the soybean seeds are soaked in an aqueous solution of theherbicide. Commonly the herbicide is present in the solution in aconcentration of approximately 2 to 6 percent by weight, and mostpreferably in a concentration of approximately 2.5 percent by weight.These concentrations have been found to be effective for the herbicideglyphosate. Concentrations for treatments with other herbicides can bedetermined empirically. Commonly the soybean seeds are simply immersedor suspended in the liquid comprising the herbicide. The soaking ofsoybean seeds is conducted for a period of time that is at leastsufficient for the herbicide to reach the embryos of the soybean seeds.A soaking time of at least 6 hours has been found to yield good results.The liquid comprising the herbicide can be simply provided at roomtemperature when the soybean seeds are in contact with the liquid andare undergoing such soaking.

Following soaking in the herbicide, the resultant seeds are planted in agrowing medium (e.g., soil) and germination of the seeds is attempted toproduce soybean plants that display herbicide resistance. The herbicideresistance can be confirmed by spraying the resulting soybean plantswith the same herbicide in a concentration typically used to kill weedsgrowing in a soybean field.

Alternatively, such screening of the resulting soybean plants forherbicide resistance can include the inclusion of the herbicide in thegrowing medium where the resulting seeds are planted. Good results areobtained in a preferred embodiment when one gallon of a solutioncontaining the herbicide in a concentration of approximately 2 to 6percent by weight is added to each 4 gallons of soil. The presence ofthe herbicide in the soil helps to assure that an atypical soybean seedhaving a harder seed coat is effectively exposed to the herbicide. Ithas been found that a small proportion of the seeds following soaking ina liquid comprising a herbicide, and planting in a growth medium, willgerminate and yield soybean plants that exhibit resistance to theherbicide.

The percentage of the soybean plants that will grow normally followingsuch seed treatments has been found to vary from variety to variety.Some varieties have produced no surviving plants in tests to date. Somevarieties have produced up to approximately 1 surviving plant per 1,000seeds, and others approximately 1 surviving plant per 25,000 seeds. Theherbicide resistance of the resulting plants can be further confirmed byanother contact (e.g., by spraying) with the herbicide. A simple fieldtest kit for herbicide resistance is available from AIT Company ofIroquois, S. Dak., as well as from other sources.

A portion of a herbicide-resistant soybean plant produced following suchgermination or a descendant thereof is analyzed to confirm that themanifest herbicide resistance is not the result of genetic engineeringinvolving the insertion by man of a foreign gene that is notnaturally-occurring into the soybean plant. This preferably is done bychecking for the presence of a promoter or genetic marker sequences thatwere introduced by man when inserting a foreign gene construct forherbicide resistance into the soybean germplasm. This analysis is usedto confirm that the subject soybean plant is not a genetically-modifiedorganism and that the manifest herbicide resistance is attributable to anaturally-occurring genetic basis other than that introduced by geneticengineering. More specifically, this analysis is used to confirm thatthe resulting herbicide-resistant soybean plant or plants were notderived in some manner (e.g., by outcrossing) from a soybean plant thathas been genetically engineered for herbicide resistance.

In accordance with the process of the present invention a soybean plantis next selected in which the herbicide resistance is under geneticcontrol and in which there is no evidence of the use of geneticengineering to produce the herbicide resistance, such as the presence ofa chimeric plant promoter-bacterial gene construct for such herbicideresistance. Any suitable technique can be utilized to confirm theabsence of the use of genetic engineering to produce the herbicideresistance. For instance, a DNA-polymerase chain reaction can beutilized. In a preferred embodiment, a DNA-polymerase chain reaction iscarried out on a portion of a soybean plant leaf. This analysis can becarried out to advantage when analyzing a portion of a young growingleaf. A DNA sequence analysis can be utilized to confirm that the genefor herbicide resistance does not conform to the sequence of a foreigngene inserted into the soybean genome by genetic engineering. At thistime the DNA sequences that are relevant are those that are in commonuse in commercially-grown transgenic crops. The sequences that are inuse and are detectable by PCR screening are the sequences for thecauliflower mosaic viral 35S promoter, the figwort mosaic virus (FMV)promoter, the individual coding sequences that encode proteins that whenexpressed render the plant resistant to a particular herbicide, e.g.,the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene frompetunia enabling tolerance to glyphosate, and the chimeric NOS-NPTII-NOSgene for kanamycin resistance. The presence and expression of thepetunia EPSPS gene conferring glyphosate resistance can also be detectedby use of a specific antibody directed against the protein encoded bythis gene. The CP4 gene for glyphosate herbicide resistance when presentalso can be detected.

Also, contemplated by the instant invention are the nucleic acids whichcomprise the genes which when expressed in the soybean plant provideherbicide resistance to that plant. Once a soybean plant which exhibitsgenetically-controlled herbicide resistance that is not attributable togenetic engineering has been identified, the gene responsible for thenaturally-occurring herbicide resistance can be genetically mapped,identified, isolated, and sequenced by anyone competent in the art. See,Plant Genomes: Methods for genetic and physical mapping. J. S. Beckmannand T. C. Osborn, 1992, Kluwer Academic Publishers, Genome mapping inPlants. A. Paterson, 1996 Harcourt Brace and Co, Maize Genome mapping A.Kalinski 1996, Diane Publishing Co., and Methods in Molecular BiologyVol. 82: Arabidopsis Protocols J. M. Martinez-Zapater and J. Salinas,1998 Humana Press. The isolated nucleic acid encoding the geneconferring the naturally-occurring herbicide resistance encodes aprotein responsible for causing the plant to be herbicide resistant.This isolated nucleic acid can then be used to (1) identify othernucleic acids which may contain naturally-occurring mutations thatprovide herbicide resistance to soybean plants; (2) introduce theisolated nucleic acid into a soybean plant which lacks herbicideresistance by genetic engineering techniques which are known to anartisan of ordinary skill; (3) insert the isolated nucleic acid into asuitable vector which can be expressed in a soybean plant; and (4)insert the vector into a plant cell (e.g., a soybean plant cell).

The present invention also contemplates the fabrication of DNAconstructs comprising the isolated nucleic acid sequence containing thecoding sequence from the gene that confers herbicide resistanceoperatively linked to plant gene expression control sequences. “DNAconstructs” are defined herein to be constructed (not-naturallyoccurring) DNA molecules useful for introducing DNA into host cells, andthe term includes chimeric genes, expression cassettes, and vectors.

As used herein “operatively linked” refers to the linking of DNAsequences (including the order of the sequences, the orientation of thesequences, and the relative spacing of the various sequences) in such amanner that the encoded protein is expressed. Methods of operativelylinking expression control sequences to coding sequences are well knownin the art. See, e.g., Maniatis et al., Molecular cloning: A LaboratoryManual. Cold Spring Harbor, N.Y. (1982), Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1989).

“Expression control sequences” are DNA sequences involved in any way inthe control of transcription or translation. Suitable expression controlsequences and methods of making and using them are known in the art.

The expression control sequences must include a promoter. The promotermay be inducible or constitutive. It may be naturally-occurring, may becomposed of portions of various naturally-occurring promoters, or may bepartially or totally synthetic. Guidance for the design of promoters isprovided by studies of promoter structure, such as that of Harley andReynolds, Nucleic Acids Res., 15, 2343-61 (1987). Also, the location ofthe promoter relative to the transcription start may be optimized. See,e.g, Roberts et al., Proc. Natl. Acad Sci. USA, 76, 760-4 (1979). Manysuitable promoters for use in plants are well known in the art.

For instance, suitable constitutive promoters for use in plants include:the promoters of plant viruses, such as the peanut chlorotic streakcaulimovirus (PCISV) promoter (U.S. Pat. No. 5,850,019), the 35S and 19Spromoter from cauliflower mosaic virus (CaMV) (Odell et al., 1313:3810-812, 1985), promoters of the Chlorella virus methyltransferasegenes (U.S. Pat. No. 5,563,328), and the full-length transcript promoterfrom figwort mosaic virus (FMV) (U.S. Pat. No. 5,378,619); the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)), ubiquitin (Christiansen et al., Plant Mol. Biol. 12:619-632(1989)), and (Christiansen et al., Plant Mol. Biol. 18: 675-689 (1992)),pEMU (Last et al., Theor. Appl Genet. 81:581-588 (1991)), MAS (Velten etal., Embo J. 3:2723-2730 (1984)), maize H# histone (Lepetit et al., Mol.Gen. Genet. 231:276-285 (1992) and Atanassova et al., Plant Journal2:291-300 (1992)), Brassica napus ALS3 (International Publication No. WO97/41228); and promoters of various Agrobacterium genes (See U.S. Pat.Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

Suitable inducible promoters for use in plants include: the promoterfrom the ACE1 system which responds to copper (Mett et al., PNAS90:4567-4571 (1993)): the promoter of the maize In2 gene which respondsto benzenesulfonomide herbicide safeners (U.S. Pat. No. 5,364,780 andGatz et al Mol. Gen. Genet. 243:32-38 (1994)), and the promoter of theTet repressor from Tn10 (Gatz et al Mol. Gen. Genet. 227:229-237(1991)). A particularly preferred promoter for use in plants is one thatresponds to an inducing agent to which plants normally do not respond.An exemplary inducible promoter of this type is the inducible promoterfrom a steroid hormone gene, the transcriptional activity of which isinduced by a glucosteroid hormone (Schena et al., PNAS 88:10421 (1991))or the recent application of a chimeric transcription activator, XVE,for use in an estrogen receptor-based inducible plant expression systemactivated by estradiol (Zou et al., The Plant Journal 24 265-273(2000)). Other inducible promoters for use in plants are described inEuropean Application No. 332104, and International Publication Nos. WO93/21334 and WO 97/06269.

Finally, promoters composed of portions of other promoters and partiallyor totally synthetic promoters can be used. See, e.g., Ni et al., PlantJournal 7:661-676 (1995) and International Publication No. WO 95/14098describing such promoters for use in plants.

The promoter may include, or be modified to include, one or moreenhancer elements. Preferably, the promoter will include a plurality ofenhancer elements. Promoters containing enhancer elements provide forhigher levels of transcription as compared to promoters that do notinclude them. Suitable enhancer elements for use in plants include thePC1SV enhancer element (U.S. Pat. No. 5,850,019), the CaMV35S enhancerelement (U.S. Pat. Nos. 5,106,739 and 5,164,316) and the FMV enhancerelement (Maiti et al., Transgenic Research 6:143-156 (1997)). See also,International Publication No. WO 96/23898 and Enhancers and EukaryoticExpression (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1983).

For efficient expression, the coding sequences are preferably alsooperatively linked to a 3′ untranslated sequence. The 3′ untranslatedsequence will include a transcription termination sequence and apolyadenylation sequence. The 3′ untranslated region can be obtainedfrom the flanking regions of genes from Agrobacterium, plant viruses,plants and other eukaryotes. Suitable 3′ untranslated sequences for usein plants include those of the cauliflower mosaic virus 35S gene, thephaseolin seed storage protein gene, the pea ribulose-1,5-bisphosphatecarboxylase small subunit E9 gene, the soybean 7S storage protein gene,the octopine synthase gene, and the nopaline synthase gene.

A 5′ untranslated leader sequence is also employed. The 5′ untranslatedleader sequence is the portion of an mRNA which extends from the 5′ CAPsite to the translation initiation codon. This region of the mRNA isnecessary for translation initiation in plants and plays a role in theregulation of gene expression. Suitable 5′ untranslated leader sequencefor use in plants includes those of alfalfa mosaic virus, cucumbermosaic virus coat protein gene, and tobacco mosaic virus.

The DNA construct may be a vector. The vector may contain one or morereplication systems which allow it to replicate in host cells.Self-replicating vectors include plasmids, cosmids and virus vectors.Alternatively, the vector may be an integrating vector which allows theintegration into the host cell's chromosome of the DNA sequence encodingthe herbicide resistance gene product. The vector desirably also hasunique restriction sites for the insertion of DNA sequences. If a vectordoes not have unique restriction sites it may be modified to introduceor eliminate restriction sites, to make it more suitable for furthermanipulation.

Vectors suitable for use in expressing the nucleic acids, which whenexpressed in a plant confer herbicide resistance, include but are notlimited to pMON979, pMON977, pMON886, pCaMVCN, and vectors derived fromthe tumor inducing (Ti) plasmid of Agrobacterium tumefaciens describedby Rogers et al., Meth. Enz˜mol. 153:253-77 (1987). The nucleic acid isinserted into the vector such that it is operably linked to a suitableplant active promoter. Suitable plant active promoters for use with thenucleic acids include CaMV35S, ACTIN, NOS and PCSLV promoters. Thevectors comprising the nucleic acid can be inserted into a plant cellusing a variety of known methods. For example, DNA transformation ofplant cells include but are not limited to Agrobacterium-mediated planttransformation, protoplast transformation, gene transfer into pollen,and injection into reproductive organs, injection into immature embryosand particle bombardment. These methods are described more fully in U.S.Pat. No. 5,756,290 and the references cited therein. Site-specificrecombination systems can also be employed to reduce the copy number andrandom integration of the nucleic acid into the soybean plant genome.For example, the Cre/lox system can be used to immediate loxsite-specific recombination in plant cells. This method can be found inChoi et al., Nuc. Acids Res. 28: B19 (2000) and elsewhere.

The herbicide resistance that results from the present invention isshown to be an infrequent naturally-occurring dominant genetic mutantand not the product of an introduced genetic modification or a mutationinduced by man. The process steps of the of the present invention havebeen found to enable the isolation of such naturally-occurring geneticmutants in soybean plants on a reliable basis. Such herbicide resistanceis under genetic control through the expression of one or more dominantgene pairs for herbicide resistance and can be readily transferred toother soybean varieties and lines, through the use of conventional plantbreeding. Hereafter such dominant gene pairs have been designated asNG^(Rn), where “n” can be any number (in this specification NG^(R1)through NG^(R6) have been utilized).

The herbicide resistance of the present invention can be provided intrue-breeding soybean varieties and lines as well as in Fi soybeanhybrids. When forming F₁ hybrids, the requisite genetic control isprovided in both parent plants (e.g., in cytoplasmic male sterile andrestorer parent plants). Also, soybean plants can be provided that areresistant to more than one herbicide when appropriatenaturally-occurring genes are incorporated into a single soybean plantsuch as by the use of conventional plant breeding followed by selection.The techniques used in such a plant breeding program are commonly knownto those skilled in the art and are described, in part, in the treatise“Breeding Field Crops” 1995 4^(th) Edition by J. Poehlman and D. Sleper,published by Iowa State University Press, Ames, Iowa.

Herbicide resistant soybean plants of the present invention can besprayed with herbicide at any stage of the plant life cycle withoutdeleterious results. For instance, herbicide resistant soybean plants ofthe present invention can be treated with a herbicide from the seedstage through flowering and during pod formation and filling withoutinjury. This is not always possible with genetically engineeredherbicide resistance. A longer and safer period for spraying with aherbicide is provided by this invention. Accordingly, a soybean growerwhen utilizing soybean plants of the present invention, can spray thesoybean field with herbicide whenever the need for weed control isapparent without restriction with respect to timing. This providesgreater weed control options and more flexibility to the soybean grower.

In yet another embodiment, the invention provides a method ofcontrolling weeds in a field where herbicide resistant soybean plants ofthe present invention or transgenic plants utilizing nucleic acidsequences, constructs or vectors in accordance the present invention aregrowing. The method comprises applying an effective amount of herbicide,in the preferred embodiment a glyphosate herbicide, to the field tocontrol the weeds. Methods of applying herbicides, including glyphosate,and the herbicide concentrations that are effective to control varioustypes of weeds are known. See, Crop Protection Reference (Chemical andPharmaceutical Press, Inc., New York, N.Y., 11^(th) edition 1995).

The following Examples are presented as specific illustrations of theclaimed invention. It should be understood, however, that the inventionis not limited to the specific details of the Examples.

EXAMPLES

In the following examples preferred embodiments are provided for theselection, establishment and development of gene sources fornaturally-occurring resistance in soybean germplasm to exposure to thenon-selective broad spectrum post-emergence herbicide glyphosate.

The identification of soybean germplasm within which naturally-occurringherbicide resistance genes reside involved the screening of thirty-foursoybean lines obtained from publicly-available seed stocks. The initialstep in the screening was to pre-soak individual batches of seedsrepresenting the thirty-four test lines in a 2 percent aqueous solutionof glyphosate at room temperature. The individual batches of seeds wereimmersed in this 2 percent solution for a period of six hours. Trialsindicated that this amount of time commonly was necessary to allow theherbicide to penetrate the seed coat, and to affect the embryo and hencegermination. This screening step was essential to determine how seedsresponded to the herbicide. Twelve of the varieties were present asapproximately 2,500 seeds. There were approximately 5,000 seeds of theremaining 22 varieties. Each variety was immersed in separate containersto assure variety identification and isolation. The varieties that weretested are identified in Table 1. TABLE 1 Soybean varieties tested forthe presence of glyphosate resistance. Variety Source ‘Dwight’University of Illinois - Dr. C. Nickel ‘Jack’ University of Illinois -Dr. C. Nickel ‘Macon’ University of Illinois - Dr. C. Nickel ‘Ina’University of Illinois - Dr. C. Nickel ‘Rend’ University of Illinois -Dr. C. Nickel ‘Iroquois’ University of Illinois - Dr. C. Nickel ‘Pana’University of Illinois - Dr. C. Nickel ‘Omaha’ University of Illinois -Dr. C. Nickel ‘Savoy’ University of Illinois - Dr. C. Nickel ‘Maverick’University of Illinois - Dr. C. Nickel 12 Non-pedigree lines Agri ProSeed Numbered 1-12 Ames, Iowa 12 Non-pedigree lines Sommer Brothers SeedNumbered 1-12 Perkin, Illinois

After this initial pre-soak, each lot of seeds was labeled and wasplaced in a flat (36″×18″×9″) containing a soil/peat mixture growthmedium, with the mixture being 50 percent top soil (sandy loam) and 50percent peat moss, that was provided at a depth of 9 inches. The 2percent solution of glyphosate and water that previously had been usedto soak the seeds was then poured over the seeds. A 2″-layer of thegrowth medium was spread over the seeds. Containers were placed in agreenhouse under ambient conditions and were watered to maintain thesoil mix at or near field capacity.

Germination and emergence of any seedlings was then observed for thenext three weeks. The majority of the herbicide-treated seeds failed togerminate. Of those that did germinate some died within a day or two andonly five of the thirty-two tested lines produced any plants thatsurvived the herbicide seed treatments and thus tolerated exposure tothe glyphosate herbicide. These five lines were identified as lines thatcontained glyphosate resistance genes within their germplasm and wereselected for further screening. Each of the five lines was obtained fromthe publicly released soybean varieties of the University of Illinoissoybean breeding program. Of these five lines, the ‘Dwight’ and ‘Jack’varieties were chosen for further screening using large quantities ofseeds.

To maximize the yield of glyphosate resistant individual plants foranalysis, and to maximize the likelihood of isolating an individual thathad sufficient glyphosate resistance to act as a founder for acommercially viable glyphosate resistant soybean line and gene source, adirect screen for glyphosate resistance was initiated using thepreviously-identified ‘Dwight’ and ‘Jack’ soybean varieties. Without theprior screening via soaking in a herbicide and survivor identificationsdescribed above, this procedure would have been unduly burdensome.

Three hundred pounds, or approximately 840,000 seeds from each variety,(‘Dwight’ and ‘Jack’), were planted in a five-acre block of land inLubbock County, Tex. After emergence, and growth to the secondtri-foliate leaf stage, glyphosate herbicide at the rate of one quart ofherbicide per acre was applied to the field. An additional applicationat the same rate was applied approximately 10 days later. Thirty daysafter the second application of glyphosate, surviving plants wereselected. Single-leaf samples from 346 surviving plants were collectedand sent to Bio Diagnostics Inc., River Falls, Wis., for testing by PCRfor the presence of the CaMV35S promoter, the only promoter used to dateto control expression in commercial lines of soybean plants geneticallyengineered for glyphosate resistance, and the NOS marker gene, used incommercially available genetically engineered lines to generatekanamycin resistance for use as a selectable marker during soybeantransformation. This analysis revealed that 178 plants tested free ofboth sequences indicating that these plants were not the result of anoutcross to a commercially available transgenic glyphosate resistantsoybean line.

The surviving plants, including those previously identified and tested,were exposed to a third application of glyphosate at the rate of onequart per acre in the field. In addition, as a result of delayedemergence, a large number of plants that were not exposed to the earliertreatments were exposed to the herbicide during this third treatment. Asa result, an additional 161 new plants were sampled for testing for thepresence of introduced genetic material, and of these 157 resulted innegative tests for both genes. All selected plants were further treatedwith herbicide, a 1 percent solution of glyphosate, by use of a handsprayer in order to assure effective herbicide contact. All plantssurvived this additional herbicide treatment.

Throughout the PCR based testing of plant samples taken from survivorsof the herbicide treatments random samples, along with positivecontrols, were chosen for duplicate testing in order to determine thereliability and consistency of the testing. As a result of this, 46plants were tested multiple times and remained negative for the presenceof both marker elements, the 35S promoter and the NOS sequence.

This screening of germplasm material from the ‘Dwight’ and ‘Jack’soybean varieties, identified as germplasm containing natural glyphosateresistance genes by use of the seed treatment protocol, resulted in theisolation of 405 individual soybean plants that were capable ofsurviving exposure to glyphosate at a rate of one quart per acre or a 1percent solution applied by a hand sprayer. Of these 405 individualsoybean plants unequivocally 325 tested negative for the presence ofeither the 35S promoter or the NOS marker gene by virtue of the PCRbased testing system of Bio Diagnostics Inc, River Falls, Wis. Theseplants were considered to contain within their genomes genes that whenexpressed delivered resistance to the of the glyphosate herbicide thatwas not the result of a genetic source currently present in commerciallyavailable soybean lines that was introduced into the germplasm of thetwo lines by plant transformation or other biotechnological means.

Of the 325 plants that tested negative for both the 35S promoter and theNOS marker gene only 230 had desirable growth characteristics and werekept for further testing. Each of these plants was grown to maturity andthe seed produced by each plant was harvested and was kept separate.Approximately fifteen seeds from each plant were germinated in flatscontaining a mixture of 50 percent top soil (sandy loam) and 50 percentpeat moss and the seedlings were allowed to grow to the first trifoliatestage. The seedlings were then exposed to a 2 percent solution ofglyphosate applied by hand spraying. The seedlings were then evaluatedapproximately ten days following this treatment for presence of the 35Spromoter and NOS marker gene, survival, growth characteristics, andplant morphology.

From this screening six plants exhibited the desired glyphosateresistance, absence of introduced gene sequences relating to commercialglyphosate resistance transgenes, appropriate growth characteristics,plant morphology, and level of seed production. As shown below five ofthese plants were derived from the ‘Dwight’ variety and one was derivedfrom the ‘Jack’ variety. These plants and the results of the PCR basedscreening are presented in Table 2. Each line is capable of being selfedto produce a homozygous line that is suitable for conventional genemapping and gene isolation protocols, know to those skilled in the art.Homozygous lines can be used to transfer genes responsible for thenaturally-occurring herbicide resistance into superior soybean lines bytraditional breeding strategies. Each line can be further tested for itsperformance in herbicide resistance trials. Each line has been renamedfor ease of identification as shown in Table 3. TABLE 2 Plant lines thatexpress glyphosate resistance obtained from the present invention. PCRTest for Ratio PCR Test for 35S and Resistant/ 35S and NOS - NOS -Sensitive Plant Original Glyphosate Original Progeny (maximum 15Designation Variety Resistance Plant Plants plants) 159 ‘Jack’ + Tested9 plants 13:0  negative × 2 Tested - all negative 221 ‘Dwight’ + Tested3 plants 9:4 negative × 2 Tested - all negative 240 ‘Dwight’ + Tested 3plants 11:2  negative × 2 Tested - all negative C-7 ‘Dwight’ + Tested 2plants 2:1 negative × 2 Tested - both negative 324 ‘Dwight’ + Tested 3plants 8:5 negative × 2 Tested - all negative L-14 ‘Dwight’ + Tested 9plants 11:3  negative × 2 Tested all negative

TABLE 3 Re-designation key for plant lines described in Table 2.Experimental Plant New Nomenclature 159 NatGen 1 221 NatGen 2 240 NatGen3 C-7  NatGen 4 324 NatGen 5 L-14 NatGen 6

The foregoing examples were substantially repeated while utilizing seedsof the publicly-available ‘Mitchell 450’ soybean variety as the startingmaterial. An experimental plant was selected which displayed therequisite naturally-occurring genetically-controlled glyphosateherbicide resistance that was not attributable to a foreign gene forherbicide resistance introduced by genetic engineering. Following theself-pollination of such plant the glyphosate herbicide resistancecontinued to be displayed in subsequent generations and was attributableto homogyzous gene NG^(R7)NG^(R7). This soybean line was designatedNatGen7.

On Oct. 24, 2002 a deposit of 2,500 seeds of soybean line NatGen 1 fromwhich homozygous gene NG^(R1)NG^(R1) is obtainable was made under theterms of the Budapest Treaty at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A. and hasreceived ATCC Accession No. PTA-4774.

On Apr. 29, 2004 a deposit of 2,500 seeds of soybean line NatGen 3 fromwhich homozygous gene NG^(R3)NG^(R3) is obtainable was made under theterms of the Budapest Treaty additionally was made at the samedepository and has received ATCC Accession No. PTA-5937.

On ______ a deposit of 2,500 seeds of soybean line NatGen 7 from whichhomozygous gene NG^(R7)NG^(R7) is obtainable was made under the terms ofthe Budapest Treaty was made at the same depository and has receivedATCC Accession No. ______.

Seeds from each of the above-identified deposits will be irrevocablymade available upon the grant of a patent that makes reference to thesedeposits. However, the availability of these seeds is not to beconstrued as a license to practice the claimed invention incontravention of rights granted under the authority of any government inaccordance with its patent or breeder's rights laws.

Although the invention has been described with reference to preferredembodiments, it is understood that variations and modifications may beresorted to as will be apparent to those skilled in the art. Suchvariations and modifications are to be considered within the purview andscope of the claims appended hereto.

1. A soybean plant which exhibits naturally-occurringgenetically-controlled glyphosate herbicide resistance that is notattributable to a foreign gene for herbicide resistance introduced bygenetic engineering obtained by the following process or a descendantthereof wherein the process comprises the steps of: (a) soaking maturesoybean seeds in the absence of induced mutagenesis which are notgenetically engineered for glyphosate herbicide resistance in a liquidcomprising a glyphosate herbicide for a period of time sufficient forthe herbicide to reach the embryos of the soybean seeds, (b) plantingsaid soybean seeds following said soaking of step (a) in a growingmedium and selecting at least one soybean plant that displays resistanceto said glyphosate herbicide, and (c) confirming the resistance to saidglyphosate herbicide in a soybean plant selected in step (b), whichexhibits naturally-occurring genetically-controlled glyphosate herbicideresistance that is not attributable to a foreign gene for herbicideresistance introduced by genetic engineering.
 2. A soybean plantaccording to claim 1 wherein said soaking of step (a) has a duration ofat least 6 hours.
 3. A soybean plant according to claim 1 wherein saidglyphosate herbicide of step (a) is provided in an aqueous solution in aconcentration of approximately 2 to 6 percent by weight.
 4. A soybeanplant according to claim 1 wherein the soybean seeds of step (a) whichare not genetically engineered for herbicide resistance are of the‘Dwight’ variety.
 5. A soybean plant according to claim 1 wherein thesoybean seeds of step (a) which are not genetically engineered forherbicide resistance are of the ‘Jack’ variety.
 6. A soybean plantaccording to claim 1 wherein the soybean seeds of step (a) which are notgenetically engineered for herbicide resistance are of the ‘Mitchell450’ variety.
 7. A soybean plant according to claim 1 wherein in step(b) said liquid comprising a glyphosate herbicide of step (a)additionally is added to the growing medium of step (b).
 8. A soybeanplant according to claim 1 wherein the confirmation of step (c) iscarried out on the basis of plant survival following contact with aglyphosate herbicide and the presence of a heritable gene for glyphosateherbicide resistance that is not attributable to a foreign gene forherbicide resistance introduced by genetic engineering.
 9. A soybeanplant according to claim 1 wherein step (b) is carried out by plantingat least 5,000 soybean seeds following said soaking of step (a) in agrowth medium, spraying the soybean plants that are produced with aglyphosate herbicide in a concentration sufficient to kill soybeanplants and weeds that lack glyphosate herbicide resistance, andconfirming the existence of a surviving soybean plant havinggenetically-controlled glyphosate herbicide resistance that is notattributable to a foreign gene for herbicide resistance introduced bygenetic engineering.
 10. A soybean plant according to claim 1 whereinsaid step (c) includes analyzing a portion of at least one soybean plantproduced in step (b) or a descendant thereof to confirm the absence of aforeign gene for herbicide resistance introduced by genetic engineering.11. A soybean plant according to claim 10 wherein the portion of thesoybean plant that is analyzed in step (c) is from a leaf.
 12. A soybeanplant according to claim 10 wherein said analysis confirms the absenceof a CaMV35S promoter, an ACTIN promoter, a NOS promoter, and a PCSLVpromoter.
 13. A soybean plant according to claim 10 wherein saidanalysis confirms the absence of the Petunia EPSPS gene for glyphosateherbicide resistance.
 14. A soybean plant according to claim 10 whereinsaid analysis of confirms the absence of the CP4 gene for glyphosateherbicide resistance.
 15. A soybean plant according to claim 1 whereinthe soybean plant of step (c) is a progeny of the at least one soybeanplant selected in step (b).
 16. A soybean plant which exhibitsnaturally-occurring genetically-controlled glyphosate herbicideresistance that is not attributable to a foreign gene for herbicideresistance introduced by genetic engineering obtained by the followingprocess or a descendant thereof wherein the process comprises the stepsof: (a) soaking mature soybean seeds in the absence of inducedmutagenesis which are not genetically engineered for glyphosateherbicide resistance selected from the ‘Dwight’ variety or the ‘Jack’variety in a liquid comprising a glyphosate herbicide for a period oftime sufficient for the herbicide to reach the embryos of the soybeanseeds, (b) planting said soybean seeds following said soaking of step(a) in a growing medium and selecting at least one soybean plantproduced upon the germination of said soybean seeds that displaysgenetically-controlled herbicide resistance following contact with saidglyphosate herbicide that is not attributable to a foreign gene forherbicide resistance introduced by genetic engineering, and (c)optionally selecting a descendant plant from at least one selectedsoybean plant of step (b) that displays genetically-controlledglyphosate herbicide resistance.
 17. A soybean seed capable of forming asoybean plant having genetically-controlled glyphosate herbicideresistance that is attributable to the homozygous gene pairNG^(R1)NG^(R1) obtainable from soybean line NatGen 1 having ATCCAccession No. PTA-4774 or its descendants.
 18. A soybean seed capable offorming a soybean plant having genetically-controlled glyphosateherbicide resistance that is attributable to the homozygous gene pairNG^(R3)NG^(R3) obtainable from soybean line NatGen 3 having ATCCAccession No. PTA-5937 or its descendants.
 19. A soybean seed capable offorming a soybean plant having genetically-controlled glyphosateherbicide resistance that is attributable to the homozygous gene pairNG^(R7)NG^(R7) obtainable from soybean line NatGen 7 having ATCCAccession No. ______ or its descendants.
 20. A soybean seed which upongermination is capable of forming a soybean plant that exhibitsnaturally-occurring genetically-controlled glyphosate herbicideresistance that is not attributable to a foreign gene for herbicideresistance introduced by genetic engineering and is obtained from theplant of claim
 1. 21. A soybean seed which upon germination is capableof forming a soybean plant that exhibits naturally-occurringgenetically-controlled herbicide resistance that is not attributable toa foreign gene for herbicide resistance introduced by geneticengineering and is obtained from the plant of claim
 16. 22. A method tocontrol weeds in a field of soybean plants according to claim 1 whereina glyphosate herbicide is applied to the field at a rate and amountsuitable for effective weed control while maintaining the viability ofsaid soybean plants.